Methods for the detection and characterization of specific nucleic acid sequences and sequence variations have been used to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection and to detect the presence of variants or alleles of genes associated with diseases, conditions, and cancers. These methods also find application in the identification of sources of nucleic acids such as in forensic analysis or for paternity determinations.
Various methods are known in the art that may be used to detect and characterize specific nucleic acid sequences and sequence variants. Nonetheless, with the completion of the nucleic acid sequencing of the human genome, as well as the genomes of numerous pathogenic organisms, the demand for fast, reliable, cost-effective and user-friendly tests for the detection of specific nucleic acid sequences continues to grow. Importantly, these tests must be able to create a detectable signal from samples that contain very few copies of the sequence of interest.
Currently available technologies include signal amplification technologies including, the polymerase chain reaction (PCR) (as described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, the disclosures of which are hereby incorporated by reference), the ligase chain reaction (LCR) (sometimes referred to as “Ligase Amplification Reaction” [LAR] described by Barany, Proc. Natl. Acad. Sci., 88:189 [1991]; Barany, PCR Methods and Applic., 1:5 [1991]; and Wu and Wallace, Genomics 4:560 [1989]), and the self-sustained sequence replication reaction (3SR) (See e.g., Guatelli et al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc. Natl. Acad. Sci., 87:7797 [1990]; Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177 [1989]; and Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). Other available technologies include direct detection technologies for quantitative detection of sequences, including the cycling probe reaction (CPR) (Duck et al., BioTech., 9:142 [1990]) and the use of branched DNA (bDNA), described by Urdea et al., Gene 61:253-264 (1987). While these techniques have been useful in certain contexts, improved systems and methods are needed to allow fast, reliable, cost-effective detection of nucleic acids without requiring inordinate amounts of sample.